The formation of polysaccharide-protein conjugates is of theoretical and practical interest (Oppenheimer, S. B., Alvarez, M. and Nnoli, J., 2008, Acta Histochemica, 110, 6-13, Ed.), (Seeberger, P. H. and Werz, D. B., 2007, Nature, 446, 1046-51, Ed.), (Bertozzi, C. R. and Kiessling L. L., 2001, Science, 29, 2357-64, Ed.) and is of particular interest in the synthesis of such conjugates for the production of carbohydrate vaccines. Carbohydrate vaccines for 26 diseases are currently being produced or under development (Astronomo, R. D. and Burton, D. R., 2010, Nat. Rev. Drug Discov., 9, 308-24, Ed.). What all these carbohydrate vaccines have in common is that the active immunogenic component is a polysaccharide-protein conjugate in which polysaccharide antigen from a pathogenic organism is coupled to a carrier protein. One of most commonly used carrier proteins for conjugation is Cross Reactive Material 197 (CRM197), a non-toxic mutant of diphtheria toxin protein, tetanus toxoid, diphtheria toxin and tetanus toxin development (Astronomo, R. D. and Burton, D. R., 2010, Nat. Rev. Drug Discov., 9, 308-24, Ed.). The most commonly used polysaccharides are Streptococcus pneumoniae polysaccharide (PnPs), Haemophilus influenze polysaccharide, Salmonella polysaccharide, Shigella polysaccharide and Neisseria meningitides polysaccharide (Astronomo, R. D. and Burton, D. R., 2010, Nat. Rev. Drug Discov., 9, 308-24, Ed.), (Tan, L. K., Carlone, G. M. and Borrow, R., 2010, New Engl. J. Med., 362, 1511-20, Ed.). The covalent attachment of polysaccharide to a carrier protein in an efficient and cost-effective manner has proved challenging (Gildersleeve, J. C., Oyelaran, O., Simpson, J. T., and Allred, B., 2008, Bioconjugate Chemistry, 19, 1485-90, Ed.) and as a result the cost of carbohydrate vaccines greatly limits their use in developing countries (Astronomo, R. D. and Burton, D. R., 2010, Nat. Rev. Drug Discov., 9, 308-24, Ed.).
The most widely used methodology for making carbohydrate-protein conjugates for vaccines is reductive amination (Gildersleeve, J. C., Oyelaran, O., Simpson, J. T., and Allred, B., 2008, Bioconjugate Chem., 19, 1485-90, Ed.), (FIG. 1). In this procedure, the non-reducing polysaccharide is activated by the introduction of aldehyde groups by reaction with periodate (Kristiansen, K. A., Potthast, A. and Christensen, B. E., 2010, Carbohyd. Res., 345, 1264-1271, Ed.), (Perlin, A. S., 2006, 60, Adv. Carbohyd. Chem. Bi., 183-250, Ed.). The activated polysaccharide is then purified and mixed with the carrier protein in an aqueous solution. In this aqueous solution, reaction between the amino groups on the protein and aldehyde groups on the polysaccharide forms an unstable imine linkage between the protein and polysaccharide. Under aqueous conditions, reduction of the imine with cyanoborohydride (FIG. 1, reaction 3) is necessary to form a stable covalent linkage between the protein and the polysaccharide.
Several factors make the coupling of polysaccharide to proteins by aqueous reductive amination problematic. In water, the concentration of imine available for reduction by cyanoborohydride is very low for several reasons: 1) Water reacts with the aldehyde to form a lactol and the equilibrium between the lactol and free aldehyde on the activated carbohydrate (FIG. 1, reaction 4) favours the lactol thus greatly reducing the concentration of free aldehyde available for reaction with the amines on the protein to form imines. 2) The deprotonated form of the amine is required for reaction with the aldehyde, however at pH 8-9 only a small fraction of the amine groups are deprotonated. 3) The amount of iminium ion that can be formed is further reduced by the high concentration of water which prevents the formation of iminium ion (FIG. 1, reaction 2). For these reasons reductive amination is slow and inefficient and requires the use of high concentrations of sugar and long reaction times (Gildersleeve, J. C., Oyelaran, O., Simpson, J. T., and Allred, B., 2008, Bioconjugate Chem., 19, 1485-90, Ed.). In addition, the reducing reaction with cynanoborohydride (FIG. 1, reaction 3) results in cyanide contamination which must be removed. Due to procedural costs and the high cost of antigenic polysaccharide, the synthesis of polysaccharide-protein conjugates by reductive amination renders carbohydrate vaccines too expensive for general use in many parts of the world (Astronomo, R. D. and Burton, D. R., 2010, Nat. Rev. Drug Discov., 9, 308-24, Ed.)
There is a need for a methodology that eliminates these difficulties and provides a straightforward and cost-efficient method for synthesizing polysaccharide-protein conjugates.